Combination of bacteriolytic therapy with magnetic field for Ehrlich tumour treatment.

Referable to the limited response of the current available cancer treatment modalities, new effective cancer fighting treatments are needed. This work investigates the efficiency of intratumoural injection of Pseudomonas aeruginosa bacteria combined with a local tumour exposure to extremely low frequency square pulsed magnetic field (ELF SPMF) in the mouse Ehrlich tumour. 64 Ehrlich ascites tumour-implanted female albino BALB/C mice were equally split up into 4 groups. Group 1 (GP1) was the positive control group. Group 2 (GP2) received a single intratumoural injection of P. aeruginosa bacteria. Group 3 (GP3) was exposed to ELF SPMF for tumour local exposure. Group 4 (GP4) was treated with P. aeruginosa intratumoural injection followed by local exposure of the tumour to ELF SPMF. Treatment monitoring was evaluated using ultrastructural examination, flow cytometry analysis in addition to the measurement of tumour dielectric properties. Tumour cell apoptosis was obvious in GP2 and GP4, but, with higher severity and percentages in GP2. Tumour biophysical properties revealed a significant increase in static conductivity σS of GP2, and decreases in dielectric increment Δɛ´of both GP2 and GP4 compared to the GP1. Unfortunately, GP2 mice showed severe signs of toxicity. We advocate the utilization of the combination of P. aeruginosa and SPMF to yield the most effective antitumour agent with less bacteria-related toxicity.

Modulation of macrophage cytokine profiles during solid tumor progression: susceptibility to Candida albicans infection.

BACKGROUND: In order to attain a better understanding of the interactions between opportunist fungi and their hosts, we investigated the cytokine profile associated with the inflammatory response to Candida albicans infection in mice with solid Ehrlich tumors of different degrees. METHODS: Groups of eight animals were inoculated intraperitoneally with 5 x 106 C. albicans 7, 14 or 21 days after tumor implantation. After 24 or 72 hours, the animals were euthanized and intraperitoneal lavage fluid was collected. Peritoneal macrophages were cultivated and the levels of IFN-gamma, TNF-alpha, IL-12, IL-10 and IL-4 released into the supernatants were measured by ELISA. Kidney, liver and spleen samples were evaluated for fungal dissemination. Tumor-free animals and animals that had only been subjected to C. albicans infection were used as control groups. RESULTS: Our results demonstrated that the mice produced more IFN-gamma and TNF-alpha and less IL-10, and also exhibited fungal clearance, at the beginning of tumor evolution. With the tumor progression, this picture changed: IL-10 production increased and IFN-gamma and TNF-alpha release decreased; furthermore, there was extensive fungal dissemination. CONCLUSION: Our results indicate that solid tumors can affect the production of macrophage cytokines and, in consequence, affect host resistance to opportunistic infections.

Modulation of macrophage cytokine profiles during solid tumor progression: susceptibility to Candida albicans infection

Background: in order to attain a better understanding of the interactions between opportunist fungi and their hosts, we investigated the cytokine profile associated with the inflammatory response to Candida albicans infection in mice with solid Ehrlich tumors of different degrees. Methods: groups of eight animals were inoculated intraperitoneally with 5 × 106 C. albicans 7, 14 or 21 days after tumor implantation. After 24 or 72 hours, the animals were euthanized and intraperitoneal lavage fluid was collected. Peritoneal macrophages were cultivated and the levels of IFN-γ, TNF-α, IL-12, IL-10 and IL-4 released into the supernatants were measured by ELISA. Kidney, liver and spleen samples were evaluated for fungal dissemination. Tumor-free animals and animals that had only been subjected to C. albicans infection were used as control groups. Results: our results demonstrated that the mice produced more IFN-γ and TNF-α and less IL-10, and also exhibited fungal clearance, at the beginning of tumor evolution. With the tumor progression, this picture changed: IL-10 production increased and IFN-γ and TNF-α release decreased; furthermore, there was extensive fungal dissemination. Conclusion: our results indicate that solid tumors can affect the production of macrophage cytokines and, in consequence, affect host resistance to opportunistic infections(AU)

Conspicuous growth of intravenously inoculated Staphylococcus aureus in subcutaneously established Ehrlich ascites tumor tissue of mice.

Intratumoral growth of Staphylococcus aureus was compared with the intrarenal growth, to examine the usefulness of the method as a marker of its pathogenicity. When 5 x 10(7) CFU/mouse of three derivatives from S. aureus Cowan I with different intrarenal growth were intravenously injected into Ehrlich tumor-bearing mice, they lodged in the tumor tissue at approximately 10(3) CFU/0.1 g by 30 min after infection, and grew in the range of 10(6) CFU/0.1 g to 10(8) CFU/0.1 g by day 4, regardless of their intrarenal growth capacity. In contrast, S. saprophyticus lodged in both tissues to the same degree as S. aureus, but did not grow at all. The time course of the staphylococcal growth was different between tumor tissue and kidney, suggesting differences in the local responses against S. aureus.

Subcutaneous growth of Staphylococcus aureus concomitantly inoculated with Ehrlich ascites tumor cells.

Intratumoral growth of Staphylococcus aureus Cowan I-derived AP332 was examined by subcutaneous inoculation of cocci in doses ranging from 18 to 1.8 x 10(5) CFU with Ehrlich ascites tumor cells. Inoculation of 18 CFU AP332 resulted in staphylococcal growth in one of five mice, and the proportion of mice established intratumoral infection increased with the initial inocula. Six other strains of S. aureus also grew in the tumor tissue, and none of the three strains of coagulase-negative staphylococci grew at all. Ethanol-killed tumor cells did not promote staphylococcal growth as vigorously as the live tumor cells, especially when the initial inoculum of AP332 was smaller than 10(4) CFU.

A novel primase-free form of murine DNA polymerase alpha induced by infection with minute virus of mice.

Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.

The role of cell population kinetics in the efficacy of penicillin--an experimental analysis and stochastic modeling of the tumour tetanus and wound tetanus of the mouse.

When investigating tetanus lethality summation curves of mice under comparable quantitative conditions following a temporarily limited administration of penicillin, the curves obtained can be calculated by the kinetics of tumour cells or wound fibroblasts. In particular, it has been shown that the optimal efficacy of penicillin, after short-time usage as compared with a long-time administration schedule, is determined by the generation time of the tetanus rods as a function of the mitotic cycle of the "pace-making" tumour cells or wound fibroblasts. Further variables of the mathematical model imply the pharmacokinetics of penicillin and the recovery process of the "hit" tetanus rods. From these results some basic experimental and clinical tetanus issues can be elucidated; thus, the mitosis theory of tetanus is being verified for the stage of incubation and of clinical manifestation, while the classical necrosis theory of the pathogenesis of tetanus infection should be valid only for the final stage.

Oncolysis by Clostridium oncolyticum M55 and subsequent enzymatic determination of sialic acid in serum.

Since the discovery of the "Clostridium tetani phenomenon", various apathogenic clostridia have been used for tumour lysis. Experiments have been conducted to achieve a tumour diagnosis using radiolabelled antibodies to clostridia. In addition, a method has been described that distinguishes, with variable success, between healthy and tumour-carrying animals by means of hemagglutination. The method outlined here uses the fact that malignant cells produce a multitude of sialic acid compounds which lie on the cell membrane and are also connected to the lipid layer of the tumour cell membrane. The apathogenic Clostridium oncolyticum M55 only germinates and multiplies in the malignant tumour tissue. Thus; bacterial hydrolases can enter the tumour tissue and lead to oncolysis. Subsequently the glycocompounds which can be detected by means of an enzymatic determination of the concentration of neuraminic acid (one of the sialic acids) in the serum are washed out into the peripheral blood. We observed these processes in mice in the Ehrlich ascites solid carcinoma and in the Lewis lung carcinoma. Using this method it was possible to detect tumour growth at an early stage with impressive accuracy. The Lewis lung carcinoma which secretes only small amounts of sialic acid glycocompounds cannot be distinguished from the control group by determination of sialic acid concentration. It was possible to detect a 52% increase in the amount of sialic acid after administration of spores of clostridia. This method makes it possible to increase the tumour marker sialic acid through manipulation of the tumour, using apathogenic clostridia, and to measure of sialic acid concentration as an indicator of the metabolic products of the tumour.

[Isolation and characteristics of endocytic vacuoles containing the influenza virus]. / Vydelenie i kharakteristika éndotsitarnykh vakuolei, soderzhashchikh virus grippa.

Endocytic vacuoles (receptosomes) containing influenza virus were isolated from the cytoplasm of Ehrlich ascitic carcinoma cells and characterized. In the sucrose density gradient, the virus-containing material was detected in two peaks with a buoyant density of 1.175-1.16 and 1.155-1.135 g/cm3 with which the activity of marker enzymes of cell plasma membranes was associated. The virus was present in receptosomes in morphologically and electrophoretically intact condition. Examinations for the lipid composition of endocytic vacuoles showed the presence in their membranes of large amounts of cholesterol and glycolipids, particularly asialo-GM1 which, according to some authors may enhance the fusion of viral and cell membranes.

Virus - tumor cell relationships. In vivo cocultivation of para-influenza type 1 (Sendai) virus and of Rous sarcoma virus (Schmidt-Ruppin strain) in mouse Ehrlich ascites carcinoma.

Co-infection of Ehrlich ascites carcinoma (EAC)-bearing mice with Sendai virus and Rous sarcoma virus (RSV) did not result in the formation of complete RSV. Sendai virus could be, however, propagated in this system over 8 serial passages. As demonstrated by immunofluorescence and complement fixation reactions, antigens specific to each virus were synthesized in EAC cells following either single or mixed virus infection. The virus progens also contained antigenic fractions incorporated from the host cell. The incomplete progens synthesized when RSV inoculation preceded that of Sendai virus possessed three polypeptide fractions characteristic of Sendai virus and one RSV-specific fraction.

Fusion between Sendai virus envelopes and biological membranes as monitored by energy transfer methods.

Chlorophyll a and chlorophyll b have been inserted into reconstituted envelopes of Sendai virus particles. Fluorescence measurements indicated a high efficiency of energy transfer between the two chlorophyll molecules due to their close proximity in the viral envelope. Fusion of reconstituted, pigmented virus envelopes with various biological cell membranes at 37 degrees C resulted in a significant decrease in the yield of energy transfer. Reduction in the efficiency of energy transfer was temperature and time dependent, and was also dependent upon the ratio between the reconstituted Sendai virus envelopes (donor) and recipient cells (acceptor). No reduction in the efficiency of energy transfer was observed when non-fusogenic, reconstituted viral envelopes were incubated with cell membranes.

[Characteristics of the gene transcription of the intracisternal A-particles]. / Osobennosti transkriptsii genov intratsisternal'nykh A-chastits.

A clone containing the gene of intracisternal A-particles (IAP-gene) was isolated by hybridization with type A double-stranded RNA from the bank of fragments of genome mouse DNA cloned in pBR322 plasmid. Using DNA fragments of this clone we studied transcription of IAP-genes in cells of MOPC-21 plasmocytoma and Ehrlich ascitic carcinoma. Transcription was shown to be partially symmetrical, with predominant transcription of one of DNA chains. The observed transcripts could not be associated with known variants of IAP-genes. The sizes of RNAs transcribed in MOPC-21 and Ehrlich ascitic carcinoma cells and their sequences differed significantly. Apparently, minor variants of IAP-genes are transcribed in the cells under study.

Tumoricidal adsorption of Staphylococcus aureus organisms on Ehrlich ascites tumor cells sensitized with rabbit antibody.

A tumoricidal effect was observed when protein A-bearing Staphylococcus aureus organisms were adsorbed on Ehrlich ascites tumor cells previously sensitized with antiserum from a rabbit immunized with Ehrlich ascites tumor cells. Electron micrographs showed that staphylococci were firmly attached to the tumor cells, which might explain how effectively the attached cocci killed the tumor cells. The tumoricidal effect was confirmed not only by an in vitro experiment but also by an in vivo one. The possible applications of the tumoricidal adsorption as an indicator for staphylococcal virulence or for selective anti-tumor action was discussed.

[Model UF-2 mutant of Staphylococcus aureus 209 P for titrating Vibrio cholera enterotoxin]. / Model' UF-2 mutanta Staphylococcus aureus 209 P dlia titrovaniia énterotoksina kholernykh vibrionov.

The identical character of the action of crude V. cholerae enterotoxin on the anaerobic dehydrogenases of the UV-2 mutant of S. aureus 209 p and the surviving culture of Ehrlich's carcinoma has been revealed. The range of this action is linked with the concentration of the toxin and varies from the stimulation of cell dehydrogenases to their complete suppression. The rapid method for the titration of the enterotoxin in the dehydrogenase suppression test with the use of the bacterial model is proposed.

Expression of vaccinia virus early mRNA in Ehrlich ascites tumor cells. 2. Part of the polysomes at an early stage of virus infection are not bound to the cytoskeleton.

A method for preparing detergent cytoskeletons from uninfected or vaccinia-virus-infected cells is described. This method resulted in the fractionation of the cytoplasmic compartment into soluble and cytoskeletal fractions. More than 85% of small molecules and tRNA were released from the cytoskeleton and recovered into the soluble fraction. The cytoskeletal fraction contained about 40% of the cytoplasmic proteins and 67% of total cytoplasmic RNA. Similar values were obtained for vaccinia-virus-infected cells. In contrast, whereas 85% of the cellular polysomes and poly(A)-rich RNA remained associated with the cytoskeleton in uninfected cells, more than 40% of vaccinia early mRNA engaged into polysomes was found to be not associated with the cytoskeleton. A similar partition was found when the viral RNA was labeled for 15 min at 5 min and 20 min after the infection or when varying the duration of the pulse. Soluble and cytoskeletal viral polysomes were found to synthesize a similar set of proteins after translation in vitro of the corresponding mRNA. The fate of rapidly labeled cellular poly(A)-rich RNA upon vaccinia virus infection was followed by a glucosamine/uridine chase procedure, and also that of relatively stable poly(A)-rich RNA after long-term labeling and chase. In both cases no release of poly(A)-rich RNA from the cytoskeleton occurred after vaccinia virus infection. These experiments reveal that cellular mRNA remains associated to the cytoskeleton in EAT cells infected with vaccinia virus (early period) whereas at least 40% of the vaccinia early polysomes are not associated with the cytoskeleton. A model for vaccinia early mRNA metabolism is presented, which may account for the rapid shut-off of host protein synthesis.

Expression of vaccinia virus early mRNA in Ehrlich ascites tumor cells. 1. Translation of cellular and viral early mRNA in cell-free systems from uninfected and virus-infected cells at the early stage.

Translation of cellular and early vaccinia RNA in nuclease-treated lysates, derived from uninfected and vaccinia-virus-infected cells at the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; this conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis l machinery from vaccinia-virus-infected cells did occur since the only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells, as suggested in the accompanying paper.

Role of gangliosides in reception of influenza virus.

The ganglioside composition of Ehrlich ascites carcinoma (EAC) cells and the role of the individual gangliosides in binding and penetration into the cell of influenza virus were determined. EAC gangliosides identical with or close to GM3, GM2, GM1, GT1a and GT1b were characterized by thin-layer chromarography, compositional analyses, methylation analysis and mass-spectrometry. The ganglioside uptake capacity of native and neuraminidase-treated EAC cells was studied with tritium-labeled gangliosides of definite structure and the binding of influenza virus to cells was determinated by using [3H]uridine-labeled virus and by hemagglutination studies. Treatment of the cells with Vibrio cholerae neuraminidase largely decreased binding of the virus. Exogenous gangliosides with a terminal galactose unit or a penultimate galactose masked by neuraminic acid were able to restore the virus-binding capacity of neuraminidase-treated cells, however, the main ganglioside of EAC cells, GM2, which carbohydrate chain is terminated by N-acetylgalactosamine, was completely ineffective. The common carbohydrate sequence of the gangliosides showing binding activity (formula; see text) is proposed to be the main recognition structure of the influenza virus receptor on the surface of EAC cells. Penetration of labeled influenza virus into the nuclei of EAC cells was evaluated by measuring the radioactivity of the nuclei of neuraminidase-treated ganglioside-loaded cells after exposition to the labeled virus. Of all gangliosides tested only trisialogangliosides of the GT1b type were able to induce increased entry of the virus into the cells and accumulation of its radioactive component into the nuclei. It is suggested that GT1b gangliosides react specifically with the virus protein responsible for membrane fusion (apparently the hemagglutinin HA2 subunit) and thus are involved in virus penetration and delivery of the virus genome to the nuclei.